Small Molecules for the Treatment of Primary Cancer and Cancer Metastasis

ABSTRACT

Certain embodiments are directed to adenosine receptor antagonists that inhibit migration and growth of cancer cells.

This Application is a continuation application of U.S. patentapplication Ser. No. 16/267,106, filed Feb. 4, 2019, which is acontinuation application of U.S. patent application Ser. No. 15/544,056,filed Jul. 17, 2017 (issued as U.S. Pat. No. 10,214,529), which is anational phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2016/013645, filed Jan. 15, 2016, which claimspriority to U.S. Provisional Application Ser. No. 62/104,705 filed Jan.17, 2015, each of which is incorporated herein by reference in itsentirety.

BACKGROUND

The bone is the most common site of metastasis in patients with advancedcancers including breast and prostate cancers (Jin et al. (2011) Int. J.Cancer 128, 2545-2561; Kohno, (2008) Int. J Clin. Oncol. 13, 18-23).Bone metastases are major, potentially fatal complications in patientswith advanced cancers. Almost all patients with skeletal metastases havesignificantly decreased quality of life due to intense pain,pathological fractures, spinal cord compression, and metaboliccomplications (Welch et al. (2003) J. Musculoskelet. Neuronal Interact.3, 30-38). In fact, post-mortem studies have shown that over 70% ofbreast cancer patients exhibited skeletal metastases, and only 20% ofthese patients are still alive five years after the discovery of themetastases (Roodman (2004) N. Engl. J Med 350, 1655-1664; Welch et al.(2003) J. Musculoskelet. Neuronal Interact. 3, 30-38). The high affinitythat cancer has for bone is explained by the “seed-and-soil hypothesis”,which was proposed over a century ago (Paget (1889) Lancet 1, 571-573).It reveals that bone tissues are preferred sites of cancer metastasisdue to their microenvironment, which provides a fertile setting in whichtumor cells can grow. Many features, such as increased blood flow aswell as the release of growth factors from cells in the bone matrix,account for the frequency of bone metastases (van der Pluijm et al.(2001) J. Bone Miner. Res. 16, 1077-1091). Thus far, the criticalfactors and mechanisms responsible for bone metastases are largelyunknown.

Bisphosphonate drugs are used to treat bone cancer metastasis and resultin decreased tumor growth, reduced bone destruction, and reduced pain(Brown and Guise (2007) Cur. Osteopor. Rep. 5, 120-127). Bisphosphonatetherapy is associated with adverse side effects, which include atrialfibrillation; arthralgia and osteonecrosis of the jaw; and ophthalmic,dermatologic and renal complications; as well as medication-inducedfractures (Junquera et al. (2009) Am. J. Otolaryngol. 30, 390-395;Truong et al. (2010) J. Am. Acad. Dermatol. 62, 672-676). Despiteadvances in the diagnosis and treatment of bone metastasis from solidtumors, the mechanism of how bisphosphonate treatment inhibits bonemetastasis at the molecular level remains to be established.

Adenosine receptor antagonist analog compounds can be used for treatmentof cancer (WO2014074529). However, there still remains a need foradditional non-hydrolysable ATP analog compounds and adenosine receptorantagonists.

SUMMARY

Certain embodiments are directed to non-hydrolysable ATP analogs thatinhibit migration and growth of cancer cells. The term non-hydrolysableATP analog refers to an ATP analog that is not effectively hydrolyzed byATPase, i.e., the analog is hydrolyzed, if at all, at a rate that isless than 5, 1, or 0.1% of the rate of ATP hydrolysis by ATPase. Certainembodiments are directed to various chemical analogs of thenon-hydrolysable ATP analog adenosine 5′-[γ-thio]triphosphate (ATPγS).These chemicals inhibit cancer cell migration and growth. Certainembodiments are directed to chemical analogs of the non-hydrolysableadenosine ATP analog 5′-[γ-thio]triphosphate (ATPγS) having the generalformula of Formula: I, including compounds P1-P6 (Table 1)

where R₁ and R₂ are selected independently from hydrogen (H), cyano(CN), C1 to C3 alkyl, halogen (fluoro (F), chloro (Cl), bromo (Br), oriodo (I)), or a trifluoromethyl (CF₃). In certain aspects R1 is selectedfrom hydrogen, cyano, C1 to C3 alkyl, halogen (fluoro (F), chloro (Cl),bromo (Br), or iodo (I)), or a trifluoromethyl, and R2 is hydrogen orfluoro. In a further aspect R1 is cyano and R2 is H, R1 is H and R2 isH, R1 is trifluoromethyl and R2 is H, R1 is fluoro and R2 is H, R1 ismethyl and R2 is H, and R1 is fluoro and R2 is fluoro.

Certain embodiments are directed to administration of one or morecompounds of Formula Ito treat cancer. The compounds can be administeredalone or in combination with other anti-cancer therapies.

Adenosine exposure can promote cancer cell growth and migration, andadenosine is produced by the metabolism of ATP. Certain embodiments aredirected to a number of chemical analogs of adenosine receptorantagonist 8-Ethoxy-9-ethyl-9H-purin-6-amine (ANR94, A2A antogonist).These compounds are inhibitors of cancer cell migration and growth. Incertain aspects the chemical analogs of adenosine receptor antagonist8-Ethoxy-9-ethyl-9H-purin-6-amine have a general formula of Formula II,including compounds P7-P10 (Table 1)

Certain aspects are directed to compounds of Formula II, where R₃ isselected from dihalomethyl, C3 to C5 cycloalkyl, or tetrahydrofuran. Incertain aspects R3 is difluoromethyl, cyclopropyl, cyclobutyl, orβ-tetrahydrofuran.

Certain embodiments are directed to administration of one or morecompounds having a formula of Formula II to treat cancer. The compoundscan be administered alone or in combination with compounds with FormulaI and/or other anti-cancer therapies.

In certain aspects one or more compounds having a formula of Formula Iand/or Formula II are administered to a subject in need of ananti-cancer treatment. In certain aspects the compounds of Formula Iand/or Formula II are administered within 1, 5, 10, 20, 30, or 60minutes or hours of each other. In a further aspect the compounds areadministered concurrently. In another aspect one or more compounds ofFormula I are administered before, during, or after administration ofone or more compounds of Formula II.

TABLE 1 List of representative compounds. Compound Code Structure P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

In certain aspects a subject or patient has bladder, blood, bone, bonemarrow, brain, breast, colorectal, esophagus, gastrointestinal, head,kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin,stomach, testicular, tongue, or uterine cancer. In a further aspect thecancer is a lung, breast, or prostate cancer. In particular aspects thecancer is a metastatic cancer, such as a bone metastasis. In certainaspects the cancer is identified as being at risk for or having apropensity for metastasis or there is no indication that the cancer hasyet metastasized. In certain aspects identification of a cancer at riskof metastasis is based on assessment of a tumor biopsy.

In certain embodiments bisphosphonate drugs can be explicitly excludedfrom the claimed invention due to their potential in vivo toxicity.

As used herein, an “inhibitor” can be a chemical compound that canreduce the activity or function of a protein. An inhibitor, for example,can inhibit directly or indirectly the activity of a protein. Directinhibition can be accomplished, for example, by binding to a protein andthereby preventing the activity of the protein, or by inhibiting anenzymatic or other activity of the protein competitively,non-competitively, or uncompetitively. Indirect inhibition can beaccomplished, for example, by binding to a protein's intended target,such as a receptor or binding partner, thereby blocking or reducingactivity of the protein.

The term “effective amount” means an amount effective, at dosages andfor periods of time necessary, to achieve the desired therapeutic orprophylactic result. An “effective amount” of an anti-cancer agent inreference to decreasing cancer cell growth or migration, means an amountcapable of decreasing, to some extent, the growth of some cancer ortumor cells, or the inhibition of the ability of a cancer or tumor cellto migrate or invade non-tumor tissue, such as bone. The term includesan amount capable of invoking a growth inhibitory, cytostatic, and/orcytotoxic effect, and/or apoptosis of the cancer or tumor cells.

A “therapeutically effective amount” in reference to the treatment ofcancer, means an amount capable of invoking one or more of the followingeffects: (1) inhibition, to some extent, of cancer or tumor growth,including slowing down growth or complete growth arrest; (2) reductionin the number of cancer or tumor cells; (3) reduction in tumor size; (4)inhibition (i.e., reduction, slowing down, or complete stopping) ofcancer or tumor cell infiltration into peripheral organs; (5) inhibition(i.e., reduction, slowing down, or complete stopping) of metastasis; (6)enhancement of anti-tumor immune response, which may, but is notrequired to, result in the regression or rejection of the tumor, or (7)relief, to some extent, of one or more symptoms associated with thecancer or tumor. The therapeutically effective amount may vary accordingto factors such as the disease state, age, sex and weight of theindividual and the ability of one or more anti-cancer agents to elicit adesired response in the individual. A “therapeutically effective amount”is also one in which any toxic or detrimental effects are outweighed bythe therapeutically beneficial effects.

The phrases “treating cancer” and “treatment of cancer” mean todecrease, reduce, or inhibit the replication of cancer cells; decrease,reduce or inhibit the spread (formation of metastases) of cancer;decrease tumor size; decrease the number of tumors (i.e. reduce tumorburden); lessen or reduce the number of cancerous cells in the body;prevent recurrence of cancer after surgical removal or other anti-cancertherapies; or ameliorate or alleviate the symptoms of the disease causedby the cancer.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIG. 1. Transwell cell migration assay with MDA-MB-231 human breastcancer cells.

FIG. 2. Soft-agar Anchorage Independent Growth assay of MDA-MB-231 cells

FIG. 3. Mammary Fat Pad Xenograft Assay

DESCRIPTION

Certain embodiments are directed to compounds having a chemical formulaof Formula I, for example P1, P2, P3, P4, P4, P5, or P6 (Table 1).

Other embodiments are directed to compounds having a chemical formula ofFormula II, for example P7, P8, P9, or P10, which are chemical analogsof adenosine receptor antagonist 8-Ethoxy-9-ethyl-9H-purin-6-amine(ANR94, A2A antogonist) (Table 1). Studies have shown that all 10compounds have inhibitory effects on cell migration. In Transwell cellmigration assay of MDA-MB-231 human breast cancer cells, all 10compounds, especially P2, P3, P4, P5 and P9 showed inhibitory effects oncell migration. At 50 μM, none of the compounds exerted any toxicity tothe cell.

Soft agar assays have been performed to determine anchorage independentcell growth of MDA-MB-231 cells with compounds P1-P10 and compounds P2and P3 were found to be most effective. There was 30% and 65% decreasein cell colonies as compared to control for P2 and P3 respectively (FIG.2).

In addition, mammary fat pad xenograft assays with MDA-MB-231 cells wereperformed. MDA-MB-231 cells were xenografted in mammary fat pad of nullmice. After tumor nodules appeared, test compounds (e.g., P3) wereinjected into these mice (500 μl of a 400 μM solution). The tumor sizefor mice receiving a test compound were compared with control mice(i.e., mice administered vehicle without test compound). After 15 daysthere was more than 50% reduction in the tumor size for mice received P3compared to those that did not (FIG. 3).

In certain aspect compounds having Formula I and/or Formula II (e.g.,compounds P1-P10) can be used to inhibit proliferation and/or migrationof cancer cells. In certain aspects the cancer is a bladder, blood,bone, bone marrow, brain, breast, colorectal, esophagus,gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary,pancreas, prostate, skin, stomach, testicular, tongue, or uterinecancer. In a further aspect the cancer is breast cancer. In still afurther aspect the cancer is prostate cancer. In particular embodimentsthe cancer is metastatic cancer, e.g., cancer that has or is at risk ofmetastasizing or migrating to the bone.

In certain embodiments, the invention also provides compositionscomprising one or more compound having the chemical formula of Formula Iand/or Formula II (e.g., P1-P2) in a pharmaceutically acceptableformulation. Thus, the use of one or more compound described herein inthe preparation of a medicament is also included. Such compositions canbe used in the treatment of a variety of cancers. In certain embodimentsthe treatment is for a metastatic cancer, e.g., lung, breast, orprostate cancer.

The compounds described herein may be formulated into therapeuticcompositions in a variety of dosage forms such as, but not limited to,liquid solutions or suspensions, tablets, pills, powders, suppositories,polymeric microcapsules or microvesicles, liposomes, and injectable orinfusible solutions. The preferred form depends upon the mode ofadministration and the particular disease targeted. The compositionsalso preferably include pharmaceutically acceptable vehicles, carriers,or adjuvants, well known in the art.

Acceptable formulation components for pharmaceutical preparations arenontoxic to recipients at the dosages and concentrations employed. Inaddition to the compounds described herein, compositions may containcomponents for modifying, maintaining, or preserving, for example, thepH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption, or penetration ofthe composition. Suitable materials for formulating pharmaceuticalcompositions include, but are not limited to, amino acids (such asglycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as acetate, borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counter ions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. (seeRemington's Pharmaceutical Sciences, 18 th Ed., (A. R. Gennaro, ed.),1990, Mack Publishing Company), hereby incorporated by reference.

Formulation components are present in concentrations that are acceptableto the site of administration. Buffers are advantageously used tomaintain the composition at physiological pH or at a slightly lower pH,typically within a pH range of from about 4.0 to about 8.5, oralternatively, between about 5.0 to 8.0. Pharmaceutical compositions cancomprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH4.0-5.5, which may further include sorbitol or a suitable substitutetherefor.

The pharmaceutical composition to be used for in vivo administration istypically sterile. Sterilization may be accomplished by filtrationthrough sterile filtration membranes. If the composition is lyophilized,sterilization may be conducted either prior to or followinglyophilization and reconstitution. The composition for parenteraladministration may be stored in lyophilized form or in a solution. Incertain embodiments, parenteral compositions are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle, ora sterile pre-filled syringe ready to use for injection.

The above compositions can be administered using conventional modes ofdelivery including, but not limited to, intravenous, intraperitoneal,oral, intralymphatic, subcutaneous administration, intraarterial,intramuscular, intrapleural, intrathecal, and by perfusion through aregional catheter. Local administration to a tumor or a metastasis inquestion is also contemplated by the present invention. Whenadministering the compositions by injection, the administration may beby continuous infusion or by single or multiple boluses. For parenteraladministration, the agents may be administered in a pyrogen-free,parenterally acceptable aqueous solution comprising the desired compoundin a pharmaceutically acceptable vehicle. A particularly suitablevehicle for parenteral injection is sterile distilled water in which oneor more anti-cancer agents are formulated as a sterile, isotonicsolution, properly preserved.

Once the pharmaceutical composition of the invention has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Suchformulations may be stored either in a ready-to-use form or in a form(e.g., lyophilized) that is reconstituted prior to administration.

If desired, stabilizers that are conventionally employed inpharmaceutical compositions, such as sucrose, trehalose, or glycine, maybe used. Typically, such stabilizers will be added in minor amountsranging from, for example, about 0.1% to about 0.5% (w/v). Surfactantstabilizers, such as TWEEN®-20 or TWEEN®-80 (ICI Americas, Inc.,Bridgewater, N.J., USA), may also be added in conventional amounts.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents. Compositions for parental administration arealso sterile, substantially isotonic and made under GMP conditions.

For the compounds described herein, alone or as part of a pharmaceuticalcomposition, such doses are between about 0.001 mg/kg and 1 mg/kg bodyweight, preferably between about 1 and 100 μg/kg body weight, mostpreferably between 1 and 10 μg/kg body weight. In certain aspects,compounds described herein can be administered by infusion to patientsin daily dosages at rates ranging from 20, 25, 30, 35, 40 to 30, 35, 40,45, 50 μg/kg/min (including all values and ranges there between) for upto 8 hours, including 1, 2, 3, 4, 5, 6, 7, or 8 hours. Compoundsdescribed herein can be administered orally at about 1, 10, 20, 30, 40,50, 60 to 50, 60, 70, 80 90, 100 μg/kg or mg/kg of body weight per day.In certain aspects the compounds described herein can be administered atabout 0.01 to 10 mg/kg of body weight per day.

Therapeutically effective doses will be easily determined by one ofskill in the art and will depend on the severity and course of thedisease, the patient's health and response to treatment, the patient'sage, weight, height, sex, previous medical history and the judgment ofthe treating physician.

In some methods of the invention, the cancer cell is a tumor cell. Thecancer cell may be in a patient. The patient may have a solid tumor. Insuch cases, embodiments may further involve performing surgery on thepatient, such as by resecting all or part of the tumor. Compositions maybe administered to the patient before, after, or at the same time assurgery. In additional embodiments, patients may also be administereddirectly, endoscopically, intratracheally, intratumorally,intravenously, intralesionally, intramuscularly, intraperitoneally,regionally, percutaneously, topically, intrarterially, intravesically,or subcutaneously. Therapeutic compositions may be administered 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moretimes, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2,3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 months.

Methods of treating cancer may further include administering to thepatient chemotherapy or radiotherapy, which may be administered morethan one time. Chemotherapy includes, but is not limited to, cisplatin(CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, transplatinum,5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine,oxaliplatin, irinotecan, topotecan, or any analog or derivative variantthereof. Radiation therapy includes, but is not limited to, X-rayirradiation, UV-irradiation, γ-irradiation, electron-beam radiation, ormicrowaves. Moreover, a cell or a patient may be administered amicrotubule stabilizing agent, including, but not limited to, taxane, aspart of methods of the invention. It is specifically contemplated thatany of the compounds or derivatives or analogs, can be used with thesecombination therapies.

Various chemical definitions related to such compounds are provided asfollows.

As used herein, the term the term “fluro” designates —F; the term“cyano” means —CN; the term “methyl” means —CH₃; the term“difluromethyl” means —CF₂H; the term “triflurometyl” means —CF₃; theterm “cyclopropyl” means a three membered saturated cycloalkyl ring; theterm “cyclobutyl” means four membered saturated cycloalkyl ring; and theterm “β-tetrahydrofuran” means a five membered saturated heterocyclylring with O as heteroatom and is substituted at the β carbon from theheteroatom.

As used herein, the term “halo” designates —F, —Cl, —Br or —I; the term“mercapto” means —SH; the term “cyano” means —CN; the term “azido” means—N₃; and the term “hydroxy” means —OH.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a linear (i.e. unbranched) or branched carbonchain, which may be fully saturated, mono- or polyunsaturated. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Saturated alkyl groups include those having one or morecarbon-carbon double bonds (alkenyl) and those having one or morecarbon-carbon triple bonds (alkynyl). The groups, —CH₃ (Me), —CH₂CH₃(Et), —CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH₂CH₂CH₂CH₃ (n-Bu),—CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂ (iso-butyl), —C(CH₃)₃(tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl), are all non-limiting examples ofalkyl groups.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a linear or branched chain having atleast one carbon atom and at least one heteroatom selected from thegroup consisting of O, N, S, P, and Si. In certain embodiments, theheteroatoms are selected from the group consisting of O and N. Theheteroatom(s) may be placed at any interior position of the heteroalkylgroup or at the position at which the alkyl group is attached to theremainder of the molecule. Up to two heteroatoms may be consecutive. Thefollowing groups are all non-limiting examples of heteroalkyl groups:trifluoromethyl, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂OH, —CH₂OCH₃, —CH₂OCH₂CF₃,—CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃, —CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH,CH₂CH₂OC(O)CH₃, —CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The terms “cycloalkyl” and “heterocyclyl,” by themselves or incombination with other terms, means cyclic versions of “alkyl” and“heteroalkyl”, respectively. Additionally, for heterocyclyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule.

The term “aryl” means a polyunsaturated, aromatic, hydrocarbonsubstituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3rings that are fused together or linked covalently). The term“heteroaryl” refers to an aryl group that contains one to fourheteroatoms selected from N, O, and S. A heteroaryl group can beattached to the remainder of the molecule through a carbon orheteroatom. Non-limiting examples of aryl and heteroaryl groups includephenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

Various groups are described herein as substituted or unsubstituted(i.e., optionally substituted). Optionally substituted groups mayinclude one or more substituents independently selected from: halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. In certain aspects the optional substituents may be furthersubstituted with one or more substituents independently selected from:halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,carbamoyl, unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl,aryl sulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl,unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optionalsubstituents include, but are not limited to: —OH, oxo(═O), —Cl, —F, Br,C₁₋₄alkyl, phenyl, benzyl, —NH₂, —NH(C₁₋₄alkyl), —N(C₁₋₄alkyl)₂, —NO₂,—S(C₁₋₄alkyl), —SO₂(C₁₋₄alkyl), —CO₂(C₁₋₄alkyl), and —O(C₁₋₄alkyl).

The term “pharmaceutically acceptable salts,” as used herein, refers tosalts of compounds of this invention that are substantially non-toxic toliving organisms. Typical pharmaceutically acceptable salts includethose salts prepared by reaction of a compound of this invention with aninorganic or organic acid, or an organic base, depending on thesubstituents present on the compounds of the invention.

Non-limiting examples of inorganic acids which may be used to preparepharmaceutically acceptable salts include: hydrochloric acid, phosphoricacid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acidand the like. Examples of organic acids which may be used to preparepharmaceutically acceptable salts include: aliphatic mono- anddicarboxylic acids, such as oxalic acid, carbonic acid, citric acid,succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphaticand aromatic sulfuric acids and the like. Pharmaceutically acceptablesalts prepared from inorganic or organic acids thus includehydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate,sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate,p-toluenesulfonate, methanesulfonate, maleate, and the like.

Suitable pharmaceutically acceptable salts may also be formed byreacting the agents of the invention with an organic base such asmethylamine, ethylamine, ethanolamine, lysine, ornithine and the like.Pharmaceutically acceptable salts include the salts formed betweencarboxylate or sulfonate groups found on some of the compounds of thisinvention and inorganic cations, such as sodium, potassium, ammonium, orcalcium, or such organic cations as isopropylammonium,trimethylammonium, tetramethylammonium, and imidazolium.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable.

Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, Selection and Use (2002), which isincorporated herein by reference.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

I. EXAMPLES

The following examples as well as the figures are included todemonstrate preferred embodiments of the invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples or figures represent techniques discovered by theinventor to function well in the practice of the invention, and thus canbe considered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

A. Materials & Methods

Cell lines and cell cultures. MDA-MB-231 cells were grown in McCoy's 5AModified Media (Gibco) supplemented with 10% FBS (Hyclone). Py8119 cellswere grown in F12K nutrient media (Gibco) supplemented with 5% FetalClone II (Fisher Scientific). All cell lines were incubated in a 5% CO₂incubator at 37° C.

Cell migration assay. Migration assays were performed in transwellmembrane filter inserts in 24-well tissue culture plates (BD BiosciencesSan Jose, Calif., USA). The transwell membrane filter inserts contained6.5-mm diameter, 8-μm pore size, 10-nm thick polycarbonate membranes.Five-hundred microliter breast cancer cell suspensions were added to theupper side of the inserts at a density of 10×10⁴ cells/insert and 750 μlCM with or without other compounds was added to the lower wells. Cellswere incubated at 37° C. for 18-20 hr. Cells that did not migratethrough the filters were removed using cotton swabs, and cells thatmigrated through the inserts were fixed and stained with Hema 3 StatPack (Fisher Scientific). The number of migrated cells in 5 fields ofview per insert was counted under a light microscope at magnification10×.

Soft agar colony formation assay. For anchorage-independent cell growth,MDA-MB-231 cells were plated in 0.4% agarose with complete mediumsupplemented with 50 μM compound (P1 to P10) on top of a 0.8% agarosebase supplemented with complete medium. Cells were maintained for about2 weeks before staining with p-iodonitrotetrazolium violet(Sigma-Aldrich, St. Louis, Mo.). Images were captured by using a scannerand the numbers of colonies were counted.

Animals. Four-week-old female athymic nude mice (Harlan Sprague-Dawley,Indianapolis, Ind., USA) were used for the mammary fat pad injections.Four- to five-week old female C57bl/6 mice were used for the intratibialinjections. Animals were maintained under the care and supervision ofthe Laboratory Animal Research facility at the University of TexasHealth Science Center, San Antonio, Tex. The animal protocol wasapproved and monitored by the Institutional Animal Care and UseCommittee.

In vivo xenograft experiment. MDA-MB-231 cells were injectedsubcutaneously in the mammary fat pad of 4-week-old female nu/nu athymicnude mice. Each mouse received bilateral subcutaneous inoculation inboth the left and right inguinal mammary fat pad areas with 100 μl ofcell suspension containing ˜1×10⁷ cells/ml in serum-free media. Animalswere randomly assigned to 3 different groups, and solid tumors wereallowed to form up to about 5 mm³ volume before treatments began.Compound P3 400 μmol/500 μl saline, or saline as a control wasadministered intraperitoneally (IP) three times a week for 3 weeks. Thegrowth of xenograft tumors was monitored twice a week and tumor size wasmeasured with a caliper in two dimensions. Tumor volumes were calculatedwith the equation V=(L×W²)×0.5 (mm³), where L is length and W is widthof a tumor.

Statistical analysis. Unless otherwise specified in the Figure Legends,the data are presented as the mean±S.E.M. of at least threedeterminations. Asterisks indicate the degree of significant differencescompared with the controls (*, P<0.05; **, P<0.01; ***, P<0.001).One-way analysis of variance (ANOVA) and Student Newman-Keuls test wereused to compare groups using GraphPad Prism 5.04 software (GraphPad).

1. A compound that is a chemical analog of adenosine receptor antagonist8-Ethoxy-9-ethyl-9H-purin-6-amine with general formula of Formula II

where R3 is a heteromethyl, cycloalkyl, or tetrahydrofuran.
 2. Thecompound of claim 1, wherein R3 is difluoro methyl, cyclopropyl,cyclobutyl, or β-tetrahydrofuran.
 3. A method for treating a breastcancer or lung cancer patient comprising administering to the patient aneffective amount of one or more compounds of claim 1.